JP7220472B2 - Liposomes, anticancer agents, and cancer treatment kits - Google Patents

Description

TECHNICAL FIELD The present invention relates to liposomes, anticancer agents, and kits for treating cancer. This application claims priority based on Japanese Patent Application No. 2017-042228 filed in Japan on March 6, 2017, the content of which is incorporated herein.

Bladder cancer is known as a cancer with many recurrences. For this reason, as a treatment for non-muscle-invasive bladder cancer that does not invade the muscle layer, bacillus Calmette-Guerin (BCG) is administered intravesically to suppress recurrence after resection of the cancer tissue by endoscopic surgery. The standard treatment is to inject into BCG administered intravesically is believed to enter the patient's cancer cells and activate the patient's immune system to damage the cancer cells. BCG may also be used to treat carcinoma in situ other than bladder cancer.

BCG is an attenuated strain derived from Mycobacterium bovis, which is a type of acid-fast bacterium, and is also used as a preventive vaccine for tuberculosis. Mycolic acid present in the cell wall is considered to be an immunostimulatory component of acid-fast bacteria including BCG (see, for example, Non-Patent Document 1).

Mycolic acid is a cell wall functional molecule characteristic of mycobacteria, and is a long-chain fatty acid with 60 to 90 carbon atoms. Mycolic acid is a characteristic large long-chain molecular fatty acid that constitutes the cell wall of mycobacteria, including Mycobacterium tuberculosis, and its hydrophobicity contributes to the stability of the cell wall. It is

On the other hand, mycolic acid and its derivatives (sugar esters such as trehalose, glucose, glycerol, etc., including free mycolic acid) cause tuberculous lesions (granulomas) in the lungs when administered to animals as pathogenic agents of tubercle bacillus. It has also attracted attention as a virulence factor. Recently, it has been revealed that mycolic acid and its derivatives are distributed in all mycobacteria, and that their pathogenicity varies depending on the subclass and carbon chain.

Mycolic acids are known to have many subclasses such as α-mycolic acid, methoxymycolic acid, ketomycolic acid, wax ester mycolic acid, epoxymycolic acid, and dihydroxymycolic acid, which have different polarities. The mycolic acids of Mycobacterium tuberculosis and BCG bacteria consist of three subclasses: α-mycolic acid, methoxymycolic acid, and ketomycolic acid.

Korf J., et al., The Mycobacterium tuberculosis cell wall component mycolic acid elicits pathogen-associated host innate immune responses, Eur. J. Immunol., 35, 890-900, 2005.

Although BCG is recognized to exhibit strong anti-tumor properties, it is a live bacterium, and therefore may cause infection when administered to a living body. In addition, due to the problem of biohazard, management, supply transportation, disposal, etc. are complicated. On the other hand, if mycolic acid itself, which is considered to be an immunostimulatory component of acid-fast bacteria, is used, the problem of biohazard does not occur, but mycolic acid can activate the immune system even if it is directly administered to the body. Have difficulty. Therefore, clinical application of mycolic acid is unknown. Accordingly, an object of the present invention is to provide a technique for clinical application of mycolic acid.

The present invention includes the following aspects.
[1] A liposome containing a mycolic acid and a sterol compound.
[2] The liposome of [1], further containing a cationic lipid.
[3] The liposome of [1] or [2], wherein the mycolic acid comprises ketomycolic acid.
[4] The liposome according to any one of [1] to [3], which has a positive zeta potential.
[5] The liposome according to any one of [1] to [4], which has a diameter of 200 nm or less.
[6] The liposome according to any one of [1] to [5], which has a polydispersity index of 0.3 or less.
[7] An anticancer agent comprising the liposome according to any one of [1] to [6] as an active ingredient.
[8] A cancer treatment kit comprising the anticancer agent of [7] and an immune checkpoint inhibitor.

ADVANTAGE OF THE INVENTION According to this invention, the technique which clinically applies mycolic acid can be provided.

1 is a photograph showing the results of thin-layer chromatography of mycolic acid in Experimental Example 1. FIG. (a) to (c) are mass spectra showing the results of mass spectrometry of α-mycolic acid, methoxymycolic acid, and ketomycolic acid in Experimental Example 2, respectively. (a) to (d) are fluorescence micrographs of T24 cells contacted with control liposomes, α-mycolic acid-containing liposomes, methoxymycolic acid-containing liposomes, and ketomycolic acid-containing liposomes in Experimental Example 5, respectively. 10 is a graph showing the results of flow cytometry analysis of T24 cells contacted with control liposomes in Experimental Example 5. FIG. (a) to (c) are graphs showing the results of T24 cells, MBT-2 cells, and MB49 cells in Experimental Example 6, respectively. FIG. 11 is a diagram illustrating an experimental schedule of Experimental Example 7; 10 is a graph showing the results of measuring the tumor volume of mice over time in Experimental Example 7. FIG. 10 is a graph showing the results of measuring the tumor volume of mice over time in Experimental Example 8. FIG. 10 is a graph showing the results of flow cytometry in Experimental Example 10. FIG. 11 is a graph showing the results of measuring the tumor volume of mice over time in Experimental Example 11. FIG. (a) and (b) are representative photographs showing the results of immunostaining of mouse tumor tissues in Experimental Example 12. FIG. (c) is a graph showing the results of counting CD4-positive T cells that infiltrated tumor tissues of mice in Experimental Example 12. FIG. (a) and (b) are representative photographs showing the results of immunostaining of mouse tumor tissues in Experimental Example 12. FIG. (c) is a graph showing the results of counting CD8-positive T cells that infiltrated tumor tissues of mice in Experimental Example 12. FIG. FIG. 13 is a diagram illustrating an experimental schedule of Experimental Example 13; 13 is a graph showing the results of measuring the tumor volume of mice over time in Experimental Example 13. FIG. 10 is a graph showing the results of measuring the tumor volume of mice over time in Experimental Example 15. FIG. (a) to (d) are graphs showing the results of measuring the concentration of TNF released into the medium of bone marrow-derived dendritic cells in Experimental Example 16. FIG. (a) to (c) are graphs showing the results of measuring the concentration of TNF released into the culture medium of bone marrow-derived macrophages in Experimental Example 16. FIG. 10 is a graph showing the results of measuring the tumor volume of mice over time in Experimental Example 18. FIG.

[Liposome]
In one embodiment, the invention provides liposomes containing mycolic acid and sterol compounds.

As will be described later in Examples, administration of the liposome of the present embodiment to a living body can activate the immune system and damage cancer cells. Therefore, the liposomes of this embodiment can be used as anticancer agents, immunoactivators, adjuvants, and the like.

Mycolic acid is a hydrophobic molecule that forms the bacterial surface barrier of acid-fast bacteria. Difficulty activating the immune system. This is probably because mycolic acid is highly hydrophobic and has low cell affinity.

In contrast, as will be described later in Examples, the liposome of the present embodiment has high cell affinity and can efficiently deliver mycolic acid into the host cytoplasm. As a result, mycolic acids can be delivered to cancer cells, activate immunity against cancer cells, and damage cancer cells.

(Liposome)
A liposome is a type of artificial vesicle composed of amphipathic substances. Amphiphilic substances include phospholipids, glycolipids, surfactants, and the like.

Phospholipids include phosphatidylcholines (eg, dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dilauroylphosphatidylcholine (DLPC), etc.) having saturated or unsaturated fatty acids with 3 to 30 carbon atoms as constituents. , phosphatidylglycerols (e.g., dioleoylphosphatidylglycerol, dipalmitoylphosphatidylglycerol, dilauroylphosphatidylglycerol, etc.), phosphatidylethanolamines (e.g., dioleoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, dilauroylphosphatidylethanolamine etc.), phosphatidylserines (e.g., dioleoylphosphatidylserine, dipalmitoylphosphatidylserine, dilauroylphosphatidylserine, etc.), phosphatidic acids, phosphatidylinositols, cardiolipins, sphingomyelin, egg yolk lecithin, soybean lecithin, and hydrogens thereof Additives and the like are included. Among them, DOPC is suitable because it is inexpensive and has low toxicity to the living body.

Glycolipids include, for example, glyceroglycolipids such as sulfoquinovosyldiglyceride, diglycosyldiglyceride, and digalactosyldiglyceride; glycosphingolipids such as galactosylcerebroside, lactosylcerebroside, and ganglioside; Moreover, Span80, Tween80, Tween20 etc. are mentioned as surfactant. Amphiphilic substances may be used singly or in combination of two or more.

Liposomes can be produced by any conventionally known method such as sonication, reverse phase evaporation, freeze-thaw, lipid dissolution, and spray drying.

(mycolic acid)
In the liposome of the present embodiment, any mycolic acid can be used without particular limitation as long as it can activate the immune system. In general, mycolic acids are difficult to chemically synthesize. Therefore, it is preferable to use it after purifying it from an acid-fast bacterium.

There are three subclasses of α-mycolic acid, methoxymycolic acid, and ketomycolic acid in the mycolic acids of Mycobacterium tuberculosis and BCG. Non-tuberculous mycobacterial mycolic acids include α-mycolic acid, ketomycolic acid, and wax ester mycolic acid, all of which are known to exhibit potent immunopotentiating activity in the form of trehalose esters. As will be described later in Examples, ketomycolic acid is particularly preferred because it tends to have a high cancer-damaging activity in vivo. Mycolic acid may be in the form of free mycolic acid or in the form of sugar esters of mycolic acid such as trehalose monoester, trehalose diester, glucose monoester, glycerol monoester and the like. In the liposome of the present embodiment, any one of these mycolic acids may be used, or a mixture of two or more thereof may be used.

The content of mycolic acid is preferably 5 to 20% by weight, more preferably 10 to 15% by weight, based on the dry weight of the liposome.

(sterol compound)
The liposome of this embodiment contains a sterol compound. The inventors have clarified that by incorporating a sterol compound into mycolic acid-containing liposomes, it is possible to make the liposomes have a smaller particle size and to increase their stability.

Examples of sterol compounds include cholesterol, 3β-[N-(dimethylaminoethane)carbamoyl]cholesterol, N-(trimethylammonioethyl)carbamoylcholesterol and the like. Among them, cholesterol can be preferably used.

The content of the sterol compound is preferably 5-20% by weight, more preferably 10-15% by weight, based on the dry weight of the liposome.

(Zeta potential)
The liposome of the present embodiment preferably has a positive zeta potential. A positive zeta potential enhances cell affinity and facilitates delivery of mycolic acids into cells. The zeta potential of the liposome is preferably 1-20 mV, more preferably 5-20 mV. The zeta potential of liposomes can be measured, for example, by electrophoretic light scattering measurement.

Methods for making the zeta potential of liposomes positive include adding a cationic substance to the amphipathic substances constituting the liposomes. For example, cationic lipids may be added as liposome materials.

Cationic lipids include those having a cholesterol skeleton, those having a glutamic acid skeleton, quaternary ammonium salts, dendron lipids and the like.

Cationic lipids having a cholesterol skeleton include, for example, [N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC-Chol).

Examples of cationic lipids having a glutamic acid backbone include N-(α-trimethylammonioacetyl)-didodecyl-D-glutamic acid chloride (TMAG) and the like.

Examples of quaternary ammonium salts include N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium bromide (DOTMA), dimethyldioctadecylammonium bromide (DDAB), 2,3- dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propaneammonium trifluoroacetate (DOSPA), dioleoyl-D-glutamate-N-2(sperminecarboxamido)ethyl (DOGS), 1,2-dimyristoyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DMRIE) and the like.

Examples of dendron lipids include dendron lipids (types "D1", "D2", "D3", "D12", "D22", and "D32", all manufactured by Hygieia Biosciences), and the like. Not limited. Among them, dendron lipids (model "D22", manufactured by Hygeia Biosciences) are preferred.

The content of the cationic lipid is preferably 1-10% by weight, more preferably 2-5% by weight, based on the dry weight of the liposome.

(diameter)
The liposomes of this embodiment are preferably cationic and preferably have a diameter of 200 nm or less. Liposome diameter can be measured, for example, by dynamic light scattering. Liposomes with a diameter of 200 nm or less can be easily sterilized by filter sterilization. Therefore, it is easy to administer to a living body.

(polydispersity index)
The liposome of the present embodiment preferably has a polydispersity index of 0.3 or less. A small polydispersity index means that the liposomes have a uniform particle size. If the liposomes have a uniform particle size, the loss can be minimized in the case of filter sterilization or the like.

[Anticancer agent]
In one embodiment, the present invention provides an anticancer agent containing the aforementioned liposome as an active ingredient. As will be described later in Examples, administration of the anticancer agent of the present embodiment to a living body can activate the immune system and damage cancer cells. Therefore, the anticancer agent of this embodiment can also be called an immunoactivator, an adjuvant, or the like.

Unlike conventional live BCG bacteria, the anticancer agent of the present embodiment does not cause infectious diseases, and is easy to manage, supply, transport, and dispose of.

Cancers to be treated with the anticancer agent of the present embodiment include cancers to which the agent can be easily administered locally, and specific examples include bladder cancer, breast cancer, melanoma, and the like. By administering the anticancer agent of this embodiment into the bladder or locally administering it by local injection or the like, the anticancer agent of this embodiment is incorporated into cancer cells. As a result, cancer cells that have taken up the anticancer agent of this embodiment are damaged by the immune system. This allows cancer to be effectively treated.

[Cancer treatment kit]
In one embodiment, the present invention provides a cancer treatment kit comprising the above-described anticancer agent and an immune checkpoint inhibitor.

By administering the above-described anticancer agent to a patient in combination with an immune checkpoint inhibitor, the patient's immune system can be activated, and the therapeutic effect of cancer can be further improved. Examples of immune checkpoint inhibitors include, but are not limited to, anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-CTLA-4 antibodies, and the like. Examples of anti-PD-1 antibodies include nivolumab, pembrolizumab and the like. Examples of anti-CTLA-4 antibodies include ipilimumab, tremelimumab and the like.

[Other embodiments]
In one embodiment, the present invention provides liposomes for the treatment of cancer, the liposomes containing mycolic acid and sterol compounds. Here, the liposomes are preferably cationic. As mycolic acid and sterol compounds, the same compounds as those described above can be used.

In one embodiment, the present invention provides the use of liposomes for manufacturing anticancer agents, the use of liposomes containing mycolic acid and sterol compounds. Here, the liposomes are preferably cationic. As mycolic acid and sterol compounds, the same compounds as those described above can be used.

In one embodiment, the invention provides a method of treating cancer comprising administering to a patient in need of treatment an effective amount of a liposome containing a mycolic acid and a sterol compound. Here, the liposomes are preferably cationic. Cancers to be treated by the treatment method of this embodiment are the same as those described above. As the mycolic acid and sterol compounds, the same ones as those described above can be used.

In one embodiment, the present invention provides a method of treating cancer comprising administering to a patient in need thereof an effective amount of a liposome containing a mycolic acid and a sterol compound and an effective amount of an immune checkpoint inhibitor. provide a way. Here, the liposomes are preferably cationic. Cancers to be treated by the treatment method of this embodiment are the same as those described above. As the mycolic acid and sterol compounds, the same ones as those described above can be used. In addition, as the immune checkpoint inhibitor, those similar to those described above can be used.

Although the present invention will be described below with reference to experimental examples, the present invention is not limited to the following experimental examples.

[Experimental example 1]
(Purification of mycolic acids from BCG)
<<Culturing of BCG>>
Mycobacterium bovis BCG Tokyo 172 strain was surface cultured at 37° C. for 9 days using Thorton's medium. Subsequently, the medium was sterilized in an autoclave at 121° C. for 15 minutes and then centrifuged to recover heat-killed BCG bacteria.

《Extraction of crude mycolic acid》
Subsequently, 100 mL of 5N aqueous sodium hydroxide solution was added to 20 g of BCG heat-killed bacteria, and autoclaved at 121° C. for 20 minutes. After cooling, 5N hydrochloric acid was added to adjust the pH to 2. Subsequently, hexane was added, and the hexane layer was collected, washed with pure water, and dried to obtain total fatty acids. Subsequently, total fatty acids were dissolved in 1 mL chloroform and 50 mL of methanol was added to form a precipitate of crude mycolic acids which was collected.

《Purification of mycolic acid》
Subsequently, 10 mL of hexane/methanol (hexane:methanol=20:1, v/v) and 250 μL of 10% trimethylsilyldiazomethane were added to 100 mg of crude mycolic acid, and allowed to stand at room temperature for 30 minutes to methyl-esterify mycolic acid. . The resulting methyl ester mycolic acid was developed four times on a silica gel-coated glass thin layer chromatography (TLC) plate (20 x 20 cm) with a hexane/ethyl acetate solvent (hexane:ethyl acetate = 20:1, v/v). , colored with iodine.

Subsequently, three methyl ester fractions of α-mycolic acid, methoxymycolic acid and ketomycolic acid were collected. Subsequently, 3 mL of toluene, 5 mL of 5N aqueous sodium hydroxide solution, and 2 mL of ethanol were added to each of the recovered mycolic acid methyl esters, and autoclaved at 121° C. for 20 minutes. Subsequently, after cooling, the mixture was acidified with 5N hydrochloric acid, hexane was added, and the purified mycolic acid in the hexane layer was recovered.

FIG. 1 is a photograph showing the results of thin-layer chromatography of mycolic acid methyl ester developed four times. In FIG. 1, lane 1 is the result of mycolic acid methyl ester purified from Mycobacterium tuberculosis, a human tuberculosis bacterium. Lane 2 is the result of mycolic acid methyl ester purified from BCG. Lane 3 is the result of α(Alpha)-mycolic acid methyl ester purified from BCG. Also, lane 4 is the result of methoxy-mycolic acid methyl ester purified from BCG. Also, lane 5 is the result of Keto-mycolic acid methyl ester purified from BCG.

[Experimental example 2]
(Confirmation of mycolic acid subclass by mass spectrometry)
The molecular weight of each subclass of mycolic acid methyl ester purified in Experimental Example 1 was analyzed by a MALDI-TOF mass spectrometer (model "Voyager DE-STR", Applied Biosystems).

Each sample was dissolved in chloroform/methanol (chloroform:methanol=2:1, v/v) at a concentration of 1 mg/mL and applied to the sample plate as 0.75 mL droplets. 2,5-dihydroxybenzoic acid (2.5-DHB) was used as matrix. TOF ion trapping was performed in positive reflection mode and the acceleration voltage was set to 20 kV.

FIGS. 2(a) to 2(c) are mass spectra showing analytical results of α-mycolic acid, methoxymycolic acid, and ketomycolic acid, respectively. Table 1 below summarizes the results of MALDI-TOF mass spectrometry of each subclass of mycolic acids.

From the results of thin-layer chromatography and mass spectrometry, the purified mycolic acids of each subclass are α-mycolic acid with 78 carbon atoms, methoxymycolic acid with 85 carbon atoms, and ketomycolic acid with 84 and 86 carbon atoms. was confirmed.

[Experimental example 3]
(Preparation of mycolic acid-containing liposome 1)
Using α-mycolic acid, methoxymycolic acid, and ketomycolic acid purified in Experimental Example 1, liposomes of Production Examples 1 to 7 having the compositions shown in Table 2 were prepared. In Table 2, DOPC represents dioleoylphosphatidylcholine. As the cationic lipid, a dendron lipid represented by the following chemical formula (1) (model "D22", Hygieia Biosciences) was used.

Specifically, first, mycolic acid, DOPC, cholesterol and cationic lipids having the composition shown in Table 2 were mixed in a mixed solvent of chloroform/methanol (2:1, v/v) and heated at 60°C. , dissolved. The solvent was then completely removed under reduced pressure. Subsequently, the lipid layer was thinned by drying to dryness under a stream of nitrogen. Subsequently, 1 mL of phosphate buffer (PBS) was added and crushed with an ultrasonic crusher (model "Biorupter", Cosmo Bio). The resulting suspension was then centrifuged at 14,000 rpm for 30 minutes to collect the pellet. The recovered pellet was then dissolved in 500 μL of PBS.

Subsequently, freezing and thawing was repeated 10 times using liquid nitrogen. Subsequently, the obtained liquid is passed through a polycarbonate membrane (pore size 200 nm) 21 times on a heat block heated to 60° C. using an extruder device (model “Mini Extruder”, Avanti Polar Lipid), Uniform liposomes were obtained. The prepared liposomes were stored at 4°C until use.

[Experimental example 4]
(Analysis of mycolic acid-containing liposomes)
For each liposome prepared in Experimental Example 3, the diameter, polydispersity index, and zeta potential were measured using a molecular property evaluation device (model "ZEN3600", Malvern).

Table 3 shows the measurement results. In Table 3, "α" represents α-mycolic acid, "methoxy" represents methoxymycolic acid, and "keto" represents ketomycolic acid. In addition, "time after preparation" indicates how many days after preparation of liposomes the measurement was performed.

As a result, as is clear from the results of the liposomes of Production Examples 1 to 3, when the mycolic acid-containing liposomes did not contain cholesterol, it was found that the diameter increased 7 days after production rather than 2 days after production. became.

In contrast, as is clear from the results of the liposomes of Production Examples 4 to 6, when the mycolic acid-containing liposomes contain cholesterol, the diameter is stable, and the diameter can be maintained at 200 nm or less even when measured 7 days after production. became clear.

In addition, as is clear from the results of the liposomes of Production Examples 4 to 6, the liposomes containing mycolic acid, cholesterol and cationic lipid had a diameter of 200 nm or less, a positive zeta potential, and a polydispersity index of 0.3. The following became clear. A small polydispersity index means that the particle size of the liposomes is uniform.

The inventors also revealed that by changing the compositions of mycolic acid, cholesterol, and cationic lipid, the zeta potential and diameter of mycolic acid-containing liposomes can be adjusted to prepare liposomes that meet the intended purpose.

[Experimental example 5]
(Study of uptake of mycolic acid-containing liposomes into cells 1)
Liposomes of Production Examples 8 to 11 were produced in the same manner as in Experimental Example 3, except that cholesterol labeled with a fluorescent dye, NBD (Avanti Polar Lipid) was used as cholesterol. Hereinafter, the liposome of Production Example 8 will be referred to as "α-mycolic acid-containing liposome", the liposome of Production Example 9 will be referred to as "methoxymycolic acid-containing liposome", and the liposome of Production Example 10 will be referred to as "ketomycolic acid-containing liposome". The liposomes of Production Example 11 are sometimes referred to as "control liposomes".

Subsequently, each liposome was added to the medium of the human bladder cancer cell line T24 at a final concentration of 50 μg/mL, allowed to stand at 37° C. for 2 hours, and observed under a confocal microscope to confirm that the liposome was incorporated into the cells. I considered whether or not NBD fluorescence indicates liposome localization. In addition, cell nuclei were fluorescently stained with 4',6-diamidino-2-phenylindole (DAPI).

FIGS. 3(a)-(d) are fluorescence micrographs of T24 cells contacted with each liposome. FIG. 3(a) shows the results of control liposomes, FIG. 3(b) shows the results of α-mycolic acid-containing liposomes, FIG. 3(c) shows the results of methoxymycolic acid-containing liposomes, and FIG. ) shows the results for ketomycolic acid-containing liposomes. As a result, it was revealed that all liposomes were incorporated into the cytoplasm of T24 cells.

FIG. 4 is a graph showing the results of flow cytometry analysis of T24 cells (with liposomes) contacted with control liposomes. For comparison, T24 cells not contacted with liposomes (no liposomes) were similarly analyzed. As a result, a cell peak showing the fluorescence of the control liposome was observed, confirming that the control liposome was taken up by the T24 cells.

[Experimental example 6]
(Examination of cytotoxic activity of mycolic acid-containing liposomes)
α-Mycolic acid-containing liposomes, methoxymycolic acid-containing liposomes, ketomycolic acid-containing liposomes and control liposomes prepared in the same manner as in Experimental Example 5 were added to human bladder cancer cell line T24 and mouse bladder cancer cells at a final concentration of 50 µg/mL. It was added to the medium of strain MBT-2 and mouse bladder cancer cell strain MB49, respectively, and examined for cytotoxic activity.

Specifically, PBS, control liposomes, or each mycolic acid-containing liposome was added to a serum-free medium, and viable cell counts were measured 24 hours and 48 hours later. The number of viable cells was measured using a kit (Dojindo Laboratories) using WST-8, a water-soluble tetrazolium salt.

5(a) to (c) are graphs showing the results of measuring the number of viable cells. FIG. 5(a) shows the results for T24 cells, FIG. 5(b) shows the results for MBT-2 cells, and FIG. 5(c) shows the results for MB49 cells. 5(a) to (c), "PBS" indicates the results of adding PBS, "control" indicates the results of adding control liposomes, and "α" indicates the results of adding α-mycolic acid-containing liposomes. , "methoxy" indicates the results of adding methoxymycolic acid-containing liposomes, and "keto" indicates the results of adding ketomycolic acid-containing liposomes.

As a result, it was found that none of the liposomes had almost any cytotoxic activity in vitro.

[Experimental example 7]
(Examination of antitumor effect of mycolic acid-containing liposomes using mouse subcutaneous tumor model 1)
Antitumor effects of mycolic acid-containing liposomes were investigated using mouse subcutaneous tumor models. FIG. 6 is a diagram explaining an experiment schedule. First, on the first day of the experiment (day 0), 0.44 mg of α-mycolic acid-containing liposomes, methoxymycolic acid-containing liposomes or ketomycolic acid-containing liposomes prepared in the same manner as in Experimental Example 5, 0.5 mg of control liposomes, or 0.5 mg of PBS .1 mL each was mixed with 500,000 mouse bladder cancer cell line MB49 and administered subcutaneously on the dorsal side of C57BL/6J mice (n=5 or 6). Tumor volumes were then measured over time. In addition, on the 3rd, 5th and 7th days after the start of the experiment, only each reagent (α-mycolic acid-containing liposomes, methoxymycolic acid-containing liposomes, ketomycolic acid-containing liposomes, prepared in the same manner as in Experimental Example 5, Control liposomes or PBS) were each additionally administered subcutaneously at the same site.

FIG. 7 is a graph showing the results of measuring tumor volume over time. In FIG. 7, "PBS" indicates the results of the PBS-administered group, "control" indicates the results of the control liposome-administered group, "α" indicates the results of the α-mycolic acid-containing liposome-administered group, and "methoxy" The results for the methoxymycolic acid-containing liposome administration group are shown, and "Keto" shows the results for the ketomycolic acid-containing liposome administration group.

As a result, on the 14th day after the start of the experiment, a significant difference in tumor volume was observed between the PBS-administered group and the ketomycolic acid-containing liposome-administered group (p=0.0067). A significant difference in tumor volume was also observed between the PBS-administered group and the methoxymycolic acid-containing liposome-administered group (p=0.012). A significant difference in tumor volume was also observed between the control liposome-administered group and the ketomycolic acid-containing liposome-administered group (p=0.0044).

Moreover, on the 21st day after the start of the experiment, a significant difference in tumor volume was observed between the PBS-administered group and the ketomycolic acid-containing liposome-administered group (p=0.014). A significant difference in tumor volume was also observed between the PBS-administered group and the methoxymycolic acid-containing liposome-administered group (p=0.037).

Moreover, on the 28th day after the start of the experiment, a significant difference in tumor volume was observed between the PBS-administered group and the ketomycolic acid-containing liposome-administered group (p=0.014).

From these results, as shown in Experimental Example 6, it was revealed that mycolic acid-containing liposomes do not have direct cytotoxic activity, but exhibit cytotoxic activity (antitumor activity) under conditions involving the immune system. . In addition, ketomycolic acid was found to exhibit the highest cytotoxic activity.

[Experimental example 8]
(Examination of the antitumor effect of mycolic acid-containing liposomes using a mouse subcutaneous tumor model 2)
The antitumor effect (antitumor activity) of mycolic acid-containing liposomes was examined using a mouse subcutaneous tumor model in the same manner as in Experimental Example 7, except that Beige mice were used instead of C57BL/6J mice. The Beige mouse is known as a model animal for Chediak-Higashi syndrome and lacks NK cells.

FIG. 8 is a graph showing the results of measuring tumor volume over time. In FIG. 8, "PBS" indicates the results of the PBS-administered group, "control" indicates the results of the control liposome-administered group, "α" indicates the results of the α-mycolic acid-containing liposome-administered group, and "methoxy" is The results for the methoxymycolic acid-containing liposome administration group are shown, and "Keto" shows the results for the ketomycolic acid-containing liposome administration group.

As a result, on the 24th day after the start of the experiment, a significant difference in tumor volume was observed between the PBS-administered group and the methoxymycolic acid-containing liposome-administered group (p=0.018). A significant difference in tumor volume was also observed between the PBS-administered group and the α-mycolic acid-containing liposome-administered group (p=0.045). A significant difference in tumor volume was also observed between the PBS-administered group and the ketomycolic acid-containing liposome-administered group (p=0.045).

From this result, it was considered that NK cells are less involved in the antitumor effect of mycolic acid-containing liposomes.

[Experimental example 9]
(Preparation of mycolic acid-containing liposomes 2)
Production Example 12 with the composition shown in Table 5 was prepared in the same manner as in Experimental Example 3 except that a dendron lipid represented by the following chemical formula (2) (model "D12", Hygeia Biosciences) was used as the cationic lipid. A liposome (hereinafter sometimes referred to as "D12 liposome") was prepared. In Table 5, DOPC represents dioleoylphosphatidylcholine.

Subsequently, the diameter, polydispersity index and zeta potential of the D12 liposomes were measured using a molecular characterization device (model "ZEN3600", Malvern). Table 6 shows the measurement results. The measurement was performed 11 days after the preparation of the liposomes. As a result, the diameter, polydispersity index, and zeta potential of the D12 liposomes were each obtained using the dendron lipid (type "D22", Hygeia Biosciences, Inc.) represented by the above chemical formula (1) as the cationic lipid. 6 liposomes (hereinafter sometimes referred to as "D22 liposomes").

[Experimental example 10]
(Study of uptake of mycolic acid-containing liposomes into cells 2)
A liposome of Production Example 13 having the composition shown in Table 7 below was prepared in the same manner as in Experimental Example 9 except that cholesterol labeled with a fluorescent dye, NBD (Avanti Polar Lipid) was used as cholesterol. In Table 7, DOPC represents dioleoylphosphatidylcholine.

For comparison, a liposome prepared in the same manner as in Production Example 10 of Experimental Example 5 (hereinafter sometimes referred to as "D22 liposome") was used. Table 7 also shows the composition of the liposomes of Preparation 10.

Subsequently, each liposome was added to the medium of mouse bladder cancer cell line MB49 at a final concentration of 50 μg/mL, allowed to stand at 37° C. for 2 hours, and analyzed by flow cytometry. FIG. 9 is a graph showing the results of flow cytometry. For comparison, MB49 cells not contacted with liposomes were similarly analyzed. In FIG. 9, "none" is the result of MB49 cells not contacted with liposomes, "D12" is the result of D12 liposomes, and "D22" is the result of D22 liposomes. As a result, it was confirmed that the D22 and D12 liposomes had the same degree of fluorescent dye development, and that the D12 liposomes were taken up by MB49 cells to the same extent as the D22 liposomes.

[Experimental example 11]
(Examination of the antitumor effect of mycolic acid-containing liposomes in a mouse subcutaneous tumor model 3)
When the dendron lipid represented by the above chemical formula (1) (model "D22", Hygieia Biosciences) is used as the cationic lipid to be contained in the mycolic acid-containing liposome, and the dendron represented by the above chemical formula (2) is used. The anti-tumor effect was examined when a lipid (type "D12", Hygeia Biosciences) was used.

Specifically, the liposomes of Production Example 12 prepared in the same manner as in Experimental Example 9 (hereinafter sometimes referred to as "D12 liposomes"), the liposomes prepared in the same manner as in Production Example 6 of Experimental Example 3 (hereinafter referred to as Sometimes referred to as "D22 liposomes"), liposomes prepared in the same manner as in Production Example 7 of Experimental Example 3 (hereinafter sometimes referred to as "control liposomes"), and phosphate buffer (PBS) It was administered to the mouse subcutaneous tumor model similar to 7, and the antitumor effect was examined. Table 8 below shows the composition of each liposome. In Table 8, DOPC represents dioleoylphosphatidylcholine.

FIG. 6 is a diagram explaining an experiment schedule. First, on the first day of the experiment (day 0), 0.44 mg of D12 liposomes, 0.44 mg of D22 liposomes, 0.5 mg of control liposomes, or 0.1 mL of PBS were each mixed with 500,000 mouse bladder cancer cell line MB49. , were administered subcutaneously on the dorsal side of C57BL/6J mice (n=5 or 6). Tumor volumes were then measured over time. On the 3rd, 5th and 7th days after the start of the experiment, each reagent alone (D12 liposome, D22 liposome, control liposome or PBS) was additionally subcutaneously administered to the same site.

FIG. 10 is a graph showing the results of measuring tumor volume over time. In FIG. 10, "PBS" indicates the results of the PBS administration group, "control" indicates the results of the control liposome administration group, "D22" indicates the results of the D22 liposome administration group, and "D12" indicates the D12 liposome administration group. shows the results of

As a result, D22 liposomes using dendron lipids (model "D22", Hygieia Biosciences) as cationic lipids were better than D12 liposomes using dendron lipids (model "D12", Hygieia Biosciences) as cationic lipids. It was found that the antitumor effect was higher than

[Experimental example 12]
(Histological examination of tumor tissue)
In the mouse subcutaneous tumor model of Experimental Example 11, infiltration of T lymphocytes into the tumor tissue was examined using mice in the D22 liposome administration group and mice in the control liposome administration group on day 10 after the start of the experiment.

First, a tumor tissue excised from each mouse was fixed and embedded in paraffin to prepare a section with a thickness of 4 μm. Subsequently, immunostaining using anti-CD4 antibody and anti-CD8 antibody was performed to examine the infiltration of T lymphocytes into the tumor tissue (n=3).

Subsequently, the immunostained tissue was observed under a microscope, and regions with high infiltration of CD4-positive T cells and CD8-positive T cells were photographed. Subsequently, CD4-positive T cells and CD8-positive T cells infiltrating the tumor tissue were counted based on the micrographs taken. T-cell counts were performed three times by one observer for the same region, and the average value was statistically analyzed.

FIG. 11(a) is a representative photograph showing the results of immunostaining the tumor tissue of mice in the control liposome-administered group with an anti-CD4 antibody. FIG. 11(b) is a representative photograph showing the result of immunostaining the tumor tissue of the D22 liposome-administered mice with an anti-CD4 antibody. FIG. 11(c) is a graph showing the results of counting CD4-positive T cells in tumor tissues of mice in each group. In FIG. 11(c), "control" indicates the results of the control liposome-administered group, and "D22" indicates the results of the D22 liposome-administered group.

In addition, FIG. 12(a) is a representative photograph showing the results of immunostaining the tumor tissue of mice in the control liposome-administered group with an anti-CD8 antibody. In addition, FIG. 12(b) is a representative photograph showing the results of immunostaining the tumor tissue of the D22 liposome-administered mice with an anti-CD8 antibody. In addition, FIG. 12(c) is a graph showing the results of counting CD8-positive T cells in tumor tissues of mice in each group. In FIG. 12(c), "control" indicates the results of the control liposome-administered group, and "D22" indicates the results of the D22 liposome-administered group.

As a result of analysis by independent t-test, as shown in FIG. 11(c), no significant difference was observed in the number of CD4-positive T cells infiltrating the tumor tissue between the control liposome-administered group and the D22 liposome-administered group. In contrast, as shown in FIG. 12(c), a significant difference was observed in the number of CD8-positive T cells that infiltrated the tumor tissue between the control liposome-administered group and the D22 liposome-administered group.

These results revealed that the mice in the D22 liposome-administered group had significantly more CD8-positive T cells infiltrating the tumor tissue than the mice in the control liposome-administered group. These results also indicate that administration of D22 liposomes infiltrates CD8-positive T cells into tumor tissues and exerts an antitumor effect in a CD8-positive T-lymphocyte-dependent manner.

[Experimental example 13]
(Examination of antitumor effect of mycolic acid-containing liposomes using mouse subcutaneous tumor model 4)
In the mouse subcutaneous tumor inhibitory effect models of Experimental Examples 7, 8, and 11, the administration of mycolic acid-containing liposomes was started simultaneously with the inoculation of tumor cells. Therefore, we investigated whether or not the administration of mycolic acid-containing liposomes after tumor formation would have a tumor-suppressing effect.

FIG. 13 is a diagram explaining an experiment schedule. First, on the first day of the experiment (day 0), 500,000 mouse bladder cancer cell line MB49 were administered subcutaneously on the dorsal side of C57BL/6J mice (n=5 or 6). Subsequently, 7 days after the start of the experiment, mycolic acid-containing liposomes were subcutaneously administered to the same site 9 times in total at intervals of 1 to 2 days, and the tumor volume was measured over time.

As the mycolic acid-containing liposomes, a group was prepared in which 0.44 mg of ketomycolic acid-containing liposomes prepared in the same manner as in Experimental Example 5 was administered per animal. The dose of mycolic acid per animal was 40 μg. For comparison, a group was also prepared in which 0.5 mg of control liposomes prepared in the same manner as in Experimental Example 5 or 0.1 mL of PBS was administered per administration per animal.

FIG. 14 is a graph showing the results of measuring tumor volume over time. In FIG. 14, "PBS" indicates the results of the PBS-administered group, "Control" indicates the results of the control liposome-administered group, and "Keto" indicates the results of the ketomycolic acid-containing liposome-administered group.

As a result, on the 17th day after the start of the experiment, a significant difference in tumor volume was observed between the PBS-administered group and the ketomycolic acid-containing liposome-administered group (p=0.048). A significant difference in tumor volume was also observed between the control liposome-administered group and the ketomycolic acid-containing liposome-administered group (p=0.015).

As a result, it was clarified that the antitumor effect was observed even when the mycolic acid-containing liposomes were administered after tumor formation.

[Experimental example 14]
(Preparation of mycolic acid-containing liposomes 3)
A mycolic acid-containing liposome (hereinafter sometimes referred to as “TDM liposome”) was prepared using trehalose dimycolic acid (TDM, trehalose diester of mycolic acid) as mycolic acid.

First, BCG (Mycobacterium bovis strain BCG Tokyo 172), Mycobacterium phlei, and Rhodococcus terrae were cultured, and trehalose dimycolic acid was extracted from each cell. Refined. Hereinafter, trehalose dimycolic acid derived from each bacterium may be referred to as "BCG-TDM", "M.phlei-TDM", and "R.terrae-TDM", respectively.

Subsequently, in the same manner as in Experimental Example 3, the liposomes of Production Example 14 (hereinafter sometimes referred to as "BCG-TDM liposomes") and the liposomes of Production Example 15 (hereinafter sometimes referred to as "M. phlei-TDM liposome") and the liposome of Production Example 16 (hereinafter sometimes referred to as "R. terrae-TDM liposome"). In Table 9, DOPC represents dioleoylphosphatidylcholine. As the cationic lipid, a dendron lipid represented by the above chemical formula (1) (model "D22", manufactured by Hygeia Biosciences) was used.

Subsequently, the diameter, polydispersity index and zeta potential of each TDM posome were measured using a molecular characterization device (model "ZEN3600", Malvern). As a result, it was revealed that the diameter, polydispersity index, and zeta potential of the TDM liposomes were comparable to those of the liposomes of Production Example 6 produced in Experimental Example 3, respectively.

[Experimental example 15]
(Examination of the antitumor effect of mycolic acid-containing liposomes using a mouse subcutaneous tumor model 5)
Using the same mouse subcutaneous tumor model as in Experimental Example 13, the antitumor effect of each TDM liposome prepared in Experimental Example 14 was examined.

As the TDM liposome, the BCG-TDM liposome prepared in Experimental Example 14, M. phlei-TDM liposomes, R. Terrae-TDM liposomes were used at 0.44 mg per dose per animal. For comparison, a group was also prepared in which 0.5 mg of control liposomes prepared in the same manner as in Experimental Example 5 or 0.1 mL of PBS was administered per administration per animal.

FIG. 15 is a graph showing the results of measuring tumor volume over time. In FIG. 15, "PBS" indicates the results of the PBS-administered group, "control" indicates the results of the control liposome-administered group, "BCG" indicates the results of the BCG-TDM liposome-administered group, and "phlei" indicates the results of the M.I. The results of the phlei-TDM liposome administration group are shown, and "terra" is R. The results of the terrae-TDM liposome-administered group are shown.

As a result, on the 14th day after the start of the experiment, a significant difference in tumor volume was observed between the PBS-administered group and the BCG-TDM liposome-administered group (p=0.04). On the other hand, the PBS-administered group and the R. Between the terrae-TDM liposome-administered group, the PBS-administered group and the M. No significant difference was observed between the phlei-TDM liposome-administered group and the phlei-TDM liposome-administered group, although a tendency toward tumor suppression was observed.

Moreover, on day 18 after the start of the experiment, a significant difference in tumor volume was observed between the PBS-administered group and the BCG-TDM liposome-administered group (p=0.02). A significant difference in tumor volume was also observed between the control liposome-administered group and the BCG-TDM liposome-administered group (p=0.02).

From the above results, it was revealed that TDM liposomes also have an antitumor effect. As mycolic acids, liposomes using trehalose dimycolic acid (TDM) derived from BCG, liposomes using TDM derived from Mycobacterium phlei, and TDM derived from Rhodococcus terrae are used. Among the liposomes used, it was revealed that the liposome using BCG-derived TDM had the highest antitumor effect.

[Experimental example 16]
(Examination of Mincle-dependent cytokine production by TDM liposomes)
Macrophage-inducible C-type lectin (Mincle) is a type of C-type lectin receptor. Known Mincle ligands include injured self, fungi, and trehalose dimycolic acid (TDM) derived from Mycobacterium tuberculosis.

TDM-bound Mincle is known to associate with the FcRγ chain, perform activation signal transduction via the immunoreceptor tyrosine-based activation motif (ITAM), and induce the production of inflammatory cytokines.

In this experimental example, bone marrow-derived dendritic cells (BDMC) and bone marrow-derived macrophages (BMM) were prepared from wild-type mice or Mincle ^−/− mice and activated with TDM liposomes to induce the production of inflammatory cytokines. considered whether or not As an inflammatory cytokine, the production of Tumor Necrosis Factor (TNF) was examined.

<<Study of bone marrow-derived dendritic cells (BDMC) 1>>
Bone marrow-derived dendritic cells (BDMC) prepared from wild-type mice or Mincle ^−/− mice were seeded in 96-well plates at a cell density of 1×10 ⁵ cells/well, respectively. Subsequently, the BCG-TDM liposomes prepared in Experimental Example 14 or M. Phlei-TDM liposomes were added to a final concentration of 100 μg/mL and cultured for 48 hours. For comparison, a group to which control liposomes prepared in the same manner as in Experimental Example 5 were added to a final concentration of 100 μg/mL and a group to which nothing was added were also prepared. Subsequently, the concentration of TNF released into the medium was measured by ELISA method.

FIG. 16(a) is a graph showing the results of measuring the concentration of TNF released into the medium. In FIG. 16(a), "BCG" indicates the results of the BCG-TDM liposome addition group, and "M.phlei" indicates the results of M. phlei. Shows the results of the phlei-TDM liposome addition group, "control" shows the results of the control liposome addition group, "none" shows the results of the group to which nothing was added, and "WT" is derived from wild-type mouse bone marrow. Results for dendritic cells are shown, and “Mincle ^−/− ” represents results for bone marrow-derived dendritic cells from Mincle ^−/− mice.

As a result, BCG-TDM liposomes or M. It was found that the addition of phlei-TDM liposomes to the medium induced the production of TNF.

On the other hand, in bone marrow-derived dendritic cells derived from Mincle ^−/− mice, BCG-TDM liposomes or M. Addition of phlei-TDM liposomes to the medium did not induce TNF production.

This result indicates that cytokine production induction by addition of TDM liposomes is Mincle-dependent.

<<Study of bone marrow-derived dendritic cells (BDMC) 2>>
BCG-TDM liposomes prepared in Experimental Example 14 and M. Phlei-TDM liposomes were added to a 96-well plate at 0.3 μg/well to coat the well surface. For comparison, a group coated with 0.3 µg/well of control liposomes prepared in the same manner as in Experimental Example 5, a group coated with nothing, and 1 µg of commercially available TDM (Nacalai Tesque) were used. A group coated with /well and a group in which lipopolysaccharide (LPS) was added to the medium at a final concentration of 10 ng/mL were also prepared.

Subsequently, bone marrow-derived dendritic cells (BDMC) prepared from wild-type mice or Mincle ^−/− mice were seeded in each well at a cell density of 1×10 ⁵ cells/well and cultured for 48 hours. Subsequently, the concentration of TNF released into the medium was measured by ELISA method.

Figures 16(b) to (d) are graphs showing the results of measuring the concentration of TNF released into the medium. In FIG. 16(b), "BCG" indicates the results of the BCG-TDM liposome-coated group, and "M.phlei" indicates the results of M. phlei. The results of the phlei-TDM liposome-coated group are shown, "control" shows the results of the control liposome-coated group, and "none" shows the results of the group not coated with anything. In addition, FIG. 16(c) shows the results of the group coated with commercially available TDM. Moreover, FIG. 16(d) shows the results of the group to which LPS was added. In addition, in FIG. 16 (b) to (d), “WT” indicates the results of wild-type mouse bone marrow-derived dendritic cells, and “Mincle ^−/− ” indicates the results of Mincle ^−/− mouse bone marrow-derived dendritic cells. Show the results.

As a result, BCG-TDM liposomes or M. It was found that coating the wells with phlei-TDM liposomes induced the production of TNF. On the other hand, in bone marrow-derived dendritic cells derived from Mincle ^−/− mice, BCG-TDM liposomes or M. Coating the wells with phlei-TDM liposomes did not induce TNF production.

This result further supports that cytokine production induction of bone marrow-derived dendritic cells by TDM liposomes is Mincle-dependent.

<<Study of bone marrow-derived macrophages (BMM)>>
Bone marrow-derived macrophages (BMM) prepared from wild-type mice or Mincle ^−/− mice were seeded in 96-well plates at a cell density of 1×10 ⁵ cells/well, respectively. Subsequently, the BCG-TDM liposomes prepared in Experimental Example 14 or M. Phlei-TDM liposomes were added to a final concentration of 100 μg/mL and cultured for 48 hours. For comparison, a group to which control liposomes prepared in the same manner as in Experimental Example 5 were added to a final concentration of 100 μg/mL, a group to which nothing was added, and commercially available TDM (Nacalai Tesque) were used. A group coated with 1 μg/well and a group in which lipopolysaccharide (LPS) was added to the medium at a final concentration of 10 ng/mL were also prepared. Subsequently, the concentration of TNF released into the medium was measured by ELISA method.

Figures 17(a) to (c) are graphs showing the results of measuring the concentration of TNF released into the medium. In FIG. 17(a), "BCG" indicates the results of the BCG-TDM liposome addition group, and "M.phlei" indicates the results of M. phlei. The results for the phlei-TDM liposome-added group are shown, "Control" indicates the results for the control liposome-added group, and "None" indicates the results for the group to which nothing was added. In addition, FIG. 17(b) shows the results of the group coated with commercially available TDM. Moreover, FIG. 17(c) shows the results of the group to which LPS was added. In addition, in FIGS. 17(a) to (c), “WT” indicates the results of wild-type mouse bone marrow-derived macrophages, and “Mincle ^−/− ” indicates the results of Mincle ^−/− mouse bone marrow-derived macrophages.

As a result, in bone marrow-derived macrophages from wild-type mice, BCG-TDM liposomes or M. It was found that the addition of phlei-TDM liposomes to the medium induced the production of TNF. On the other hand, in bone marrow-derived macrophages derived from Mincle ^−/− mice, BCG-TDM liposomes or M. Addition of phlei-TDM liposomes to the medium did not induce TNF production.

This result indicates that the induction of cytokine production in bone marrow-derived macrophages by the addition of TDM liposomes is Mincle-dependent.

[Experimental example 17]
(Large-scale preparation of TDM liposomes)
A large amount of TDM liposomes of Production Example 17 having the composition shown in Table 10 was industrially prepared. BCG-TDM purified in Experimental Example 14 was used as TDM. Specifically, 15.0 mg of BCG-TDM, 15.0 mg of cholesterol, and 5.0 mg of cationic lipid (model "D22", Hygeia Biosciences) were weighed and put into a glass vial.

Subsequently, 5.4 mL of DOPC-chloroform solution (25 mg/mL) was added and thoroughly stirred at room temperature to dissolve lipid components. Subsequently, chloroform was removed by drying under reduced pressure. Subsequently, 3.0 mL of tert-butyl alcohol was added and stirred at normal temperature to dissolve the lipid component. Subsequently, 14 mL of PBS was added, and the mixture was heated and stirred in a 58° C. water bath. Subsequently, after confirming the dissolution of the lipid component, the mixture was cooled to room temperature. Subsequently, the liposomes were passed through syringe filters of 0.2 μm and 0.1 μm, and dialyzed using PBS as a dialysate to remove tert-butyl alcohol to obtain TDM liposomes of Production Example 17.

Subsequently, the diameter, polydispersity index, and zeta potential of the TDM liposomes of Production Example 17 were measured using a molecular property evaluation device (model "ZETA SIZER Nano-ZS", Malvern). Table 11 shows the measurement results. As a result, it was revealed that the diameter, polydispersity index, and zeta potential of the TDM liposomes of Production Example 17 were comparable to those of the liposomes of Production Example 6 produced in Experimental Example 3, respectively.

[Experimental example 18]
(Examination of the antitumor effect of mycolic acid-containing liposomes using a mouse subcutaneous tumor model 6)
Using the same mouse subcutaneous tumor model as in Experimental Example 7, the antitumor effect of the TDM liposome of Production Example 17 prepared in Experimental Example 17 was examined.

0.44 mg of the TDM liposome of Production Example 17 was used per administration per animal. For comparison, 0.44 mg of BCG-TDM liposome (hereinafter sometimes referred to as "TDM-D22") prepared in the same manner as in Production Example 14 of Experimental Example 14 was administered per animal. 0.44 mg of the BCG-TDM liposome of Production Example 18 (hereinafter sometimes referred to as "TDM-D12") prepared in the same manner as in Experimental Example 14 with the composition shown in Table 12 below; A group to which 0.5 mg of the control liposomes prepared as above or 0.1 mL of PBS was administered was also prepared. Table 12 below shows the composition of each liposome. In Table 12 below, "D22" represents a dendron lipid (model "D22", manufactured by Hygieia Bioscience), and "D12" represents a dendron lipid (model "D12", manufactured by Hygieia Bioscience).

FIG. 18 is a graph showing the results of measuring tumor volume over time. In FIG. 18, "PBS" indicates the results of the PBS administration group, "control" indicates the results of the control liposome administration group, "TDM-D22" indicates the results of the TDM-D22 administration group described above, and "TDM- D12" shows the results of the TDM-D12 administration group described above, and "Production Example 17" shows the results of the TDM liposome administration group of Production Example 17.

As a result, on the 25th day after the start of the experiment, a significant difference in tumor volume was observed between the PBS-administered group and the TDM-D22-administered group (p=0.02). In addition, compared with the PBS-administered group and the control liposome-administered group, the tumor volume tended to decrease in the TDM liposome-administered group of Production Example 17.

From the above results, it was confirmed that the TDM liposome of Production Example 17 exhibits an antitumor effect. Also, in TDM liposomes, it is better to use a dendron lipid (model "D22", Hygieia Bioscience) as a cationic lipid, and a case where a dendron lipid (model "D12", Hygieia Bioscience) is used as a cationic lipid. It was found that the antitumor effect was higher than

ADVANTAGE OF THE INVENTION According to this invention, the technique which clinically applies mycolic acid can be provided.

Claims (6)

A liposome containing at least one mycolic acid selected from the group consisting of α-mycolic acid, methoxymycolic acid and ketomycolic acid, cholesterol and a dendron lipid represented by the following chemical formula (1).

2. The liposome of Claim 1 , wherein the zeta potential is positive. 3. A liposome according to claim 1 or 2 , having a diameter of 200 nm or less. A liposome according to any one of claims 1 to 3 , which has a polydispersity index of 0.3 or less. An anticancer agent comprising the liposome according to any one of claims 1 to 4 as an active ingredient. A cancer treatment kit comprising the anticancer agent according to claim 5 and an immune checkpoint inhibitor.

Images